Role of Primary Afferents in Arthritis Induced Spinal Microglial Reactivity.

Increased afferent input resulting from painful injury augments the activity of central nociceptive circuits via both neuron-neuron and neuron-glia interactions. Microglia, resident immune cells of the central nervous system (CNS), play a crucial role in the pathogenesis of chronic pain. This study provides a framework for understanding how peripheral joint injury signals the CNS to engage spinal microglial responses. During the first week of monosodium iodoacetate (MIA)-induced knee joint injury in male rats, inflammatory and neuropathic pain were characterized by increased firing of peripheral joint afferents. This increased peripheral afferent activity was accompanied by increased Iba1 immunoreactivity within the spinal dorsal horn indicating microglial activation. Pharmacological silencing of C and A afferents with co-injections of QX-314 and bupivacaine, capsaicin, or flagellin prevented the development of mechanical allodynia and spinal microglial activity after MIA injection. Elevated levels of ATP in the cerebrospinal fluid (CSF) and increased expression of the ATP transporter vesicular nucleotide transporter (VNUT) in the ipsilateral spinal dorsal horn were also observed after MIA injections. Selective silencing of primary joint afferents subsequently inhibited ATP release into the CSF. Furthermore, increased spinal microglial reactivity, and alleviation of MIA-induced arthralgia with co-administration of QX-314 with bupivacaine were recapitulated in female rats. Our results demonstrate that early peripheral joint injury activates joint nociceptors, which triggers a central spinal microglial response. Elevation of ATP in the CSF, and spinal expression of VNUT suggest ATP signaling may modulate communication between sensory neurons and spinal microglia at 2 weeks of joint degeneration.

The retrotrapezoid nucleus and the neuromodulation of breathing.

Breathing is regulated by a host of arousal and sleep-wake state-dependent neuromodulators to maintain respiratory homeostasis. Modulators such as acetylcholine, norepinephrine, histamine, serotonin (5-HT), adenosine triphosphate (ATP), substance P, somatostatin, bombesin, orexin, and leptin can serve complementary or off-setting functions depending on the target cell type and signaling mechanisms engaged. Abnormalities in any of these modulatory mechanisms can destabilize breathing, suggesting that modulatory mechanisms are not overly redundant but rather work in concert to maintain stable respiratory output. The present review focuses on the modulation of a specific cluster of neurons located in the ventral medullary surface, named retrotrapezoid nucleus, that are activated by changes in tissue CO2/H+ and regulate several aspects of breathing, including inspiration and active expiration.

Hydrotropic function of ATP in the crystalline lens.

The hypothesis proposed herein is presented to explain the unexpectedly high concentration of ATP and provide evidence to support its hydrotropic function in the crystalline lens determined using 31P NMR. The lens, historically considered to be a metabolically quiescent organ, has the requisite machinery to synthesize ATP, such that the homeostatic level is maintained at about 3 mM. This relatively high concentration of ATP has been found to be consistent among multiple mammalian species including humans. This millimolar quantity is many times greater than the micromolar amounts required for the other known functions of ATP. The recent postulation that ATP at millimolar concentrations functions as a hydrotrope in various cell/tissue homogenates preventing protein aggregation coupled with observations presented herein, provide support for extending the hypothesis that ATP functions as a hydrotrope not only in homogenates but in an intact functioning organ, the crystalline lens. Concentrations of ATP of this magnitude are hypothesized to be required to maintain protein solubility and effectively prevent protein aggregation. This concept is important considering protein aggregation is the etiology for age-related cataractogenesis. ATP is a common ubiquitous intracellular molecule possessing the requisite hydrotropic properties for maintaining intracellular proteins in a fluid, non-aggregated state. It is proposed that the amphiphilic ATP molecule shields the hydrophobic regions on intralenticular fiber cell protein molecules and provides a hydrophilic interfacial surface comprised of the ATP negatively charged triphosphate side chain. Evidence is presented that this side chain is exposed to and has been reported to organize intracellular interstitial water to form an interfacial rheologically dynamic water layer. Such organization of water is substantiated with the effect of deuterium oxide (heavy water) on ATP line widths of the side chain phosphates measured ex vivo by 31P NMR. A novel model is presented to propose how this water layer separates adjacent lens fiber cell proteins, keeping them from aggregating. This hypothesis proposes that ATP can prevent protein aggregation in normal intact lenses, and with declining concentrations can be related to the disease process in age-related cataractogenesis, an affliction that affects every older human being.

Mechanism of Nucleoside Triphosphate Diphosphohydrolase-1-Associated Imbalance in Adenosine Diphosphate Degradation, B-Cell Activation, and Related Injury During Acute Antibody-Mediated Rejection.

OBJECTIVE: The objective of this study was to investigate the effect of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) during acute antibody-mediated rejection (AMR). METHODS: NTPDase1 overexpression, NTPDase1 knockout, and wild-type nude mice skin graft models were used to induce acute AMR. NTPDase1 expression in B cells, NTPDase1 messenger RNA expression in skin grafts, extracellular adenosine diphosphate (ADP) concentration, B-cell volume and surface antigens expression, average platelet transport rate, and ultrastructure and apoptosis of skin graft cells were investigated. RESULTS: During acute AMR in nude mice, higher NTPDase1 expression caused lower extracellular ADP concentration, smaller increase in B-cell volume, and major histocompatibility complex II surface antigen expression, suggesting a negative correlation between them; higher NTPDase1 expression also caused slower average platelet transport rate and less severe skin graft injury, suggesting a negative correlation between them. Pretreatment with high-dose exogenous NTPDase1 inhibited platelet activation and protected skin grafts, but it resulted in prolonged bleeding time (by 51.4%) and prolonged coagulation time (by 44.1%). CONCLUSION: An NTPDase1-associated imbalance in extracellular ADP degradation may contribute to B-cell activation, platelet activation, and more severe skin graft injury in nude mice. Pretreatment with high-dose exogenous NTPDase1 effectively protected skin grafts in nude mice at 1 week, but it increased the risk of bleeding.

Involvement of adenosine triphosphate-sensitive potassium channels in the neuroprotective activity of hydrogen sulfide in the 6-hydroxydopamine-induced animal model of Parkinson's disease.

Studies have shown that hydrogen sulfide (H2S) exerts a neuroprotective effect and may have a therapeutic value for treating neurodegenerative diseases including Parkinson's disease. However, little is known about the mechanisms underlying the neuroprotective activity of H2S in vivo. Here, we evaluated the effect of glibenclamide, an ATP-sensitive potassium channel blocker, on the neuroprotective activity of H2S in the 6-hydroxydopamine (6-OHDA) animal model of Parkinson's disease. 6-OHDA was administered by stereotaxic surgery into the medial forebrain bundle. Sodium hydrosulfate (NaHS, 3 and 5.6 mg/kg), as a donor of H2S, alone or in combination with glibenclamide (5 mg/kg), was daily injected for 7 days starting 1-2 h before the stereotaxic surgery. After an apomorphine-induced rotational test, the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta was determined by immunofluorescence. The striatal dopamine level and oxidative stress markers were also measured in brain homogenates. Pretreatment with NaHS significantly attenuated 6-OHDA-induced motor asymmetry in the rotational test. Histological and biochemical evaluations demonstrated that NaHS, especially at high dose, increased the survival of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta and reduced the decreasing effect of 6-OHDA on striatal dopamine levels. However, co-administration of glibenclamide reversed the antiparkinsonian and neuroprotective effects of NaHS. However, glibenclamide did not change the reducing effect of NaHS on 6-OHDA-induced overproduction of malondialdehyde. Our data show that ATP-sensitive potassium channels are involved in the antiparkinsonian and neuroprotective effects of H2S in the 6-OHDA animal model of Parkinson's disease.

Extracellular nucleotides as novel, underappreciated pro-metastatic factors that stimulate purinergic signaling in human lung cancer cells.

BACKGROUND: One of the challenging problems of current radio-chemotherapy is recurrence and metastasis of cancer cells that survive initial treatment. We propose that one of the unwanted effects of radiochemotherapy is the release from damaged ("leaky") cells of nucleotides such as ATP and UTP that exert pro-metastatic functions and can directly stimulate chemotaxis of cancer cells. METHODS: To address this problem in a model of human lung cancer (LC), we employed several complementary in vitro and in vivo approaches to demonstrate the role of extracellular nucleotides (EXNs) in LC cell line metastasis and tumor progression. We measured concentrations of EXNs in several organs before and after radiochemotherapy. The purinergic receptor agonists and antagonists, inhibiting all or selected subtypes of receptors, were employed in in vitro and in vivo pro-metastatic assays. RESULTS: We found that EXNs accumulate in several organs in response to radiochemotherapy, and RT-PCR analysis revealed that most of the P1 and P2 receptor subtypes are expressed in human LC cells. EXNs were found to induce chemotaxis and adhesion of LC cells, and an autocrine loop was identified that promotes the proliferation of LC cells. Most importantly, metastasis of these cells could be inhibited in immunodeficient mice in the presence of specific small molecule inhibitors of purinergic receptors. CONCLUSIONS: Based on this result, EXNs are novel pro-metastatic factors released particularly during radiochemotherapy, and inhibition of their pro-metastatic effects via purinergic signaling could become an important part of anti-metastatic treatment.

Purinergic system dysfunction in mood disorders: a key target for developing improved therapeutics.

Uric acid and purines (such as adenosine) regulate mood, sleep, activity, appetite, cognition, memory, convulsive threshold, social interaction, drive, and impulsivity. A link between purinergic dysfunction and mood disorders was first proposed a century ago. Interestingly, a recent nationwide population-based study showed elevated risk of gout in subjects with bipolar disorder (BD), and a recent meta-analysis and systematic review of placebo-controlled trials of adjuvant purinergic modulators confirmed their benefits in bipolar mania. Uric acid may modulate energy and activity levels, with higher levels associated with higher energy and BD spectrum. Several recent genetic studies suggest that the purinergic system - particularly the modulation of P1 and P2 receptor subtypes - plays a role in mood disorders, lending credence to this model. Nucleotide concentrations can be measured using brain spectroscopy, and ligands for in vivo positron emission tomography (PET) imaging of adenosine (P1) receptors have been developed, thus allowing potential target engagement studies. This review discusses the key role of the purinergic system in the pathophysiology of mood disorders. Focusing on this promising therapeutic target may lead to the development of therapies with antidepressant, mood stabilization, and cognitive effects.

Effects of flavonoid-containing preparation Extralife on hydrogen peroxide production and functioning of mitochondrial ATP-dependent potassium channel.

Extralife, a Pentaphylloides fruticos extract, in concentrations of 0.005-10 µg/ml dose-dependently increased H2O2 production in rat heart mitochondria in the presence of respiration substrates. Extralife decreased ATP-induced accumulation of H2O2 related to inhibition of mitochondrial ATP-dependent potassium channel. This effect was observed only at low doses of the adaptogen (0.05-3 µg/ml). High doses of the substance (5-10 µg/ml) did not abolish ATP-dependent production of H2O2 and increased the rate of H2O2 generation by the mitochondria. We concluded that Extralife in trace concentrations could activate mitochondrial ATP-dependent potassium channel and decrease H2O2 accumulation in the mitochondria.

MFGE8 inhibits inflammasome-induced IL-1ß production and limits postischemic cerebral injury.

Milk fat globule-EGF 8 (MFGE8) plays important, nonredundant roles in several biological processes, including apoptotic cell clearance, angiogenesis, and adaptive immunity. Several recent studies have reported a potential role for MFGE8 in regulation of the innate immune response; however, the precise mechanisms underlying this role are poorly understood. Here, we show that MFGE8 is an endogenous inhibitor of inflammasome-induced IL-1ß production. MFGE8 inhibited necrotic cell-induced and ATP-dependent IL-1ß production by macrophages through mediation of integrin ß(3) and P2X7 receptor interactions in primed cells. Itgb3 deficiency in macrophages abrogated the inhibitory effect of MFGE8 on ATP-induced IL-1ß production. In a setting of postischemic cerebral injury in mice, MFGE8 deficiency was associated with enhanced IL-1ß production and larger infarct size; the latter was abolished after treatment with IL-1 receptor antagonist. MFGE8 supplementation significantly dampened caspase-1 activation and IL-1ß production and reduced infarct size in wild-type mice, but did not limit cerebral necrosis in Il1b-, Itgb3-, or P2rx7-deficient animals. In conclusion, we demonstrated that MFGE8 regulates innate immunity through inhibition of inflammasome-induced IL-1ß production.

Energy depletion in seizures: anaplerosis as a strategy for future therapies.

Seizure activity can lead to energy failure and neuronal injury, resulting in neurological and cognitive sequelae. Moreover, mutations affecting genes encoding for proteins that maintain energy homeostasis within the cell often result in an epileptic phenotype, implying that energy failure can contribute to epileptogenesis. Indeed, there is evidence to indicate that the efficacy of the ketogenic diet, a treatment for refractory epilepsy, can be partly explained by its effect on increasing energetic substrates. The ATP level, reflecting the energy level of a cell, is maintained by the potential gradient across the mitochondrial membrane. This potential gradient is maintained by NADH/H(+) equivalents, produced by reactions within the tricarboxylic acid cycle (TCA-cycle). Anaplerosis, the replenishment of TCA-cycle substrates, therefore represents an appealing strategy to address energy failure such as occurs in seizures. There is accumulating evidence that pyruvate, a classical anaplerotic substrate, has seizure suppressive effects and protects against seizure induced cell death. This review summarizes the evidence for the contribution of TCA cycle deficits in generating seizures. We highlight the role for TCA substrate supplementation in protecting against seizures and seizure induced cell death, and propose that these are important targets for future translational research addressing energy depletion in seizures. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

Distribution of the origin of adenosine triphosphatesensitive atrial tachycardias with the earliest activation recorded in the His bundle catheter: are they limited to the immediate vicinity of the His bundle?

BACKGROUND: The aim of this study was to assess the spatial distribution of the origins of adenosine triphosphate (ATP) sensitive focal atrial tachycardias (AT) that have their earliest activation recorded in the His bundle (HB) catheter. METHODS AND RESULTS: Catheters were placed according to the standard fashion for an electrophysiologic study of supraventricular arrhythmia, namely, high right atrium, HB, coronary sinus, and right ventricle. The ATs with their earliest activation recorded in the HB catheter and that were terminated by rapid injection of ATP (4.3 ± 2.5mg), formed the study group (n=12). After catheter ablation of these ATs, the distances between the successful ablation site and the HB area were measured. Only one successful site was near the HB and the other sites were at the noncoronary sinus of Valsalva (n=6), tricuspid annulus (n=3), right atrial septum (n=1), and left atrial septum (n=1). The average distance between the HB catheter and successful site was 10.4 ± 8.8mm. In 5 of the 12 cases (the 3 tricuspid and 2 septal foci), the distances were greater than 10mm. CONCLUSIONS: When ablating ATP-sensitive AT with the earliest activation recorded in the HB catheter, it is important to perform detailed mapping not only around the HB.

Involvement of ATP in noxious stimulus-evoked release of glutamate in rat medullary dorsal horn: a microdialysis study.

Our electrophysiological studies have shown that both purinergic and glutamatergic receptors are involved in central sensitization of nociceptive neurons in the medullary dorsal horn (MDH). Here we assessed the effects of intrathecal administration of apyrase (a nucleotide degrading enzyme of endogenous adenosine 5-triphosphate [ATP]), a combination of apyrase and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, an adenosine A1 receptor antagonist), or 2,3-O-2,4,6-trinitrophenyl-adenosine triphosphate (TNP-ATP, a P2X1, P2X3, P2X2/3 receptor antagonist) on the release of glutamate in the rat MDH evoked by application of mustard oil (MO) to the molar tooth pulp. In vivo microdialysis was used to dialyse the MDH every 5 min, and included 3 basal samples, 6 samples after drug treatment and 12 samples following application of MO. Tooth pulp application of MO induced a significant increase in glutamate release in the MDH. Superfusion of apyrase or TNP-ATP alone significantly reduced the MO-induced glutamate release in the MDH, as compared to vehicle. Furthermore, the suppressive effects of apyrase on glutamate release were reduced by combining it with DPCPX. This study demonstrates that application of an inflammatory irritant to the tooth pulp induces glutamate release in the rat MDH in vivo that may be reduced by processes involving endogenous ATP and adenosine.

ATP as a mediator of erythrocyte-dependent regulation of skeletal muscle blood flow and oxygen delivery in humans.

In healthy human beings, blood flow to dynamically contracting skeletal muscle is regulated primarily to match oxygen (O(2)) delivery closely with utilisation. This occurs across a wide range of exercise intensities, as well as when exercise is combined with conditions that modify blood O(2) content. The red blood cells (RBCs), the primary O(2) carriers in the blood, contribute to the regulation of the local processes matching O(2) supply and demand. This is made possible by the ability of RBCs to release the vasoactive substance adenosine triphosphate (ATP) in response to reductions in erythrocyte and plasma O(2), as well as to other adjuvant metabolic and mechanical stimuli. The regulatory role of RBCs in human beings is supported by the observations that, i) exercising skeletal muscle blood flow responds primarily to changes in the amount of O(2) bound to the erythrocyte haemoglobin molecules, rather than the amount of O(2) in plasma, and ii) exercising muscle blood flow can almost double (from 260 to 460 ml min(-1) 100 g(-1)) with alterations in blood O(2) content, such that O(2) delivery and are kept constant. Besides falling blood O(2) content, RBCs release ATP when exposed to increased temperature, reduced pH, hypercapnia, elevated shear stress and augmented mechanical deformation, i.e. conditions that exist in the microcirculation of active skeletal muscle. ATP is an attractive mediator signal for skeletal muscle blood flow regulation, not only because it can act as a potent vasodilator, but also because of its sympatholytic properties in the human limb circulations. These properties are essential to counteract the vasoconstrictor effects of concurrent increases in muscle sympathetic nerve activity and circulating vasoconstrictor substances during exercise. Comparison of the relative vasoactive potencies and sympatholytic properties of ATP, other nucleotides, and adenosine in human limbs, suggests that intravascular ATP exerts its vasodilator and sympatholytic effects directly, and not via its degradation compounds. In conclusion, current evidence clearly indicates that RBCs are involved directly in the regulation of O(2) supply to human skeletal muscle during dynamic exercise. Further, intravascular ATP might be an important mediator in local metabolic sensing and signal transduction between the RBCs and the endothelial and smooth muscle cells in the vascular beds of skeletal muscle.

Mechanisms of ATP-mediated vasodilation in humans: modest role for nitric oxide and vasodilating prostaglandins.

ATP is an endothelium-dependent vasodilator, and findings regarding the underlying signaling mechanisms are equivocal. We sought to determine the independent and interactive roles of nitric oxide (NO) and vasodilating prostaglandins (PGs) in ATP-mediated vasodilation in young, healthy humans and determine whether any potential role was dependent on ATP dose or the timing of inhibition. In protocol 1 (n = 18), a dose-response curve to intrabrachial infusion of ATP was performed before and after both single and combined inhibition of NO synthase [N(G)-monomethyl-L-arginine (L-NMMA)] and cyclooxygenase (ketorolac). Forearm blood flow (FBF) was measured via venous occlusion plethysmography and forearm vascular conductance (FVC) was calculated. In this protocol, neither individual nor combined NO/PG inhibition had any effect on the vasodilatory response (P = 0.22-0.99). In protocol 2 (n = 16), we determined whether any possible contribution of both NO and PGs to ATP vasodilation was greater at low vs. high doses of ATP and whether inhibition during steady-state infusion of the respective dose of ATP impacted the dilation. FBF in this protocol was measured via Doppler ultrasound. In protocol 2, infusion of low (n = 8)- and high-dose (n = 8) ATP for 5 min evoked a significant increase in FVC above baseline (low = 198 ± 24%; high = 706 ± 79%). Infusion of L-NMMA and ketorolac together reduced steady-state FVC during both low- and high-dose ATP (P < 0.05), and in a subsequent trial with continuous NO/PG blockade, the vasodilator response from baseline to 5 min of steady-state infusion was similarly reduced for both low (ΔFVC = -31 ± 11%)- and high-dose ATP (ΔFVC -25 ± 11%; P = 0.70 low vs. high dose). Collectively, our findings indicate a potential modest role for NO and PGs in the vasodilatory response to exogenous ATP in the human forearm that does not appear to be dose or timing dependent; however, this is dependent on the method for assessing forearm vascular responses. Importantly, the majority of ATP-mediated vasodilation is independent of these putative endothelium-dependent pathways in humans.

[Effect of hypoxenum on bioenergetic processes in mitochondria and the activity of ATP-sensitive potassium channel].

The effect of hypoxenum on bioenergetic processes in heart and liver mitochondria of rats, connected with respiration, the generation of hydrogen peroxide, and the activity of ATP-sensitive K-channel ((mitoK)ATP) has been studied. It was shown that hypoxenum in the concentration range of 0.05-10 microg/ml stimulates respiration, increases the coupling in the respiratory chain, and enhances the formation of H2O2 and energy-dependent swelling associated with potassium transport in mitochondria. Hypoxenum removes the inhibitory effect of ATP on the energy-dependent swelling of mitochondria and partially reduces the accumulation of H2O2 in the presence of ATP. The role of antihypoxic and antioxidant action of hypoxenum associated with the activation of (mitoK)ATP is discussed.

A new approach to sperm preservation based on bioenergetic theory.

To date, attempts to preserve chicken sperm have been based on a trial-and-error experimental approach. The present work outlines the development of an alternative approach based on empiricism and bioenergetic theory. In previous work, we found fowl sperm motility to be dependent on mitochondrial calcium cycling, phospholipase A(2), and long-chain fatty acids as an endogenous energy source. It is noteworthy that fowl sperm reside within the sperm storage tubules (SST) of the oviduct over an interval of days to weeks after insemination. In this regard, a model for in vivo sperm storage was developed and tested in additional previous research. Sperm penetration of the SST, sperm residence within the SST, and sperm egress from the SST can be explained in terms mitochondrial function. Understanding sperm function and longevity in terms of bioenergetics presented the possibility that sperm could be inactivated by disrupting mitochondrial calcium cycling and could thereby be preserved. However, this possibility also posed a problem: maintenance of the inner membrane potential of the mitochondrion within inactivated sperm. This report describes a series of experiments in which fowl sperm were inactivated by treatment with the calcium chelator tetrasodium 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and then reactivated by treatment with calcium ions. The effect of tetrasodium 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid on mitochondrial calcium cycling was confirmed by flow cytometry and confocal microscopy. When treated sperm were cooled to 10 degrees C, inactivated sperm could be reactivated throughout a 5-h storage interval. When stored sperm were held for 3 h before reactivation and insemination, fertility was 88% of the control. Storage did not affect hatchability. In summary, short-term storage was realized by manipulating mitochondrial function. We propose that 1) complex V consumes ATP within inactivated sperm and, by doing so, maintains the inner membrane potential of the mitochondrion, 2) ATP is regenerated within inactivated sperm by the action of creatine kinase on phosphocreatine, and 3) necrosis follows depletion of intracellular phosphocreatine. Therefore, future attempts to preserve chicken sperm can be based on a theory that encompasses regulation of energy production, a biological context in which sperm cells are motile, and the consequences of mitochondrial failure.

Drosophila argonaute1 and argonaute2 employ distinct mechanisms for translational repression.

microRNAs induce translational repression by binding to partially complementary sites on their target mRNAs. We have established an in vitro system that recapitulates translational repression mediated by the two Drosophila Argonaute (Ago) subfamily proteins, Ago1 and Ago2. We find that Ago1-RISC (RNA-induced silencing complex) represses translation primarily by ATP-dependent shortening of the poly(A) tail of its mRNA targets. Ago1-RISC can also secondarily block a step after cap recognition. In contrast, Ago2-RISC competitively blocks the interaction of eIF4E with eIF4G and inhibits the cap function. Our finding that the two Ago proteins in flies regulate translation by different mechanisms may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis.

An adjuvant role of in situ dendritic cells (DCs) in linking innate and adaptive immunity.

Dendritic cells (DCs) work as a natural adjuvant to elicit T cell immunity. Though DCs have been widely used in immunotherapy, little is known about their number and function in patients with cancer or autoimmune disease. In recent studies, antigen has been targeted to DCs through DC-specific receptors, such as DEC205, the mannose receptor and dying cell receptors. However, antigen captured by DCs in the absence of danger signals induces tolerance. Therefore, the duration and/or magnitude of danger signals plays a crucial role in generating an immunogeneic response. Various danger signals, i.e., pathogen-associated molecular pattern (PAMP), damage-associated molecular pattern (DAMP) and the activation of innate lymphocytes, serve as maturation signals for DCs. An immunotherapeutic approach which delivers both maturation signals and antigen to DCs would link the innate and adaptive arms of the immune system for a more effective and global immune response. It is therefore crucial to determine optimal conditions for antigen delivery to DCs in an environment suited to maximally stimulate the immune system.

Drosophila ortholog of succinyl-CoA synthetase {beta} subunit: a novel modulator of Drosophila KCNQ channels.

Voltage-gated KCNQ potassium channels are responsible for slowly activating potassium currents in heart, brain, and other tissues. Functional defects of KCNQ channels are linked with many diseases, including epilepsy and cardiac arrhythmias. Therefore KCNQ potassium channels have been widely studied, especially in the CNS. We have identified Drosophila CG11963, which encodes a protein orthologous to the beta subunit of mammalian succinyl-CoA synthetase (SCS, also known as succinate thiokinase), as a novel modulator of Drosophila KCNQ channels. Direct interaction of CG11963 and dKCNQ was demonstrated by yeast two-hybrid screen and coimmunoprecipitation. Cell surface biotinylation experiments further confirmed that CG11963 resides on the plasma membrane of tsA-201 cells. Coexpression of CG11963 with dKCNQ shifts the conductance-voltage (G-V) relationship of dKCNQ channels to more positive membrane potentials in Chinese hamster ovary (CHO) cells. Moreover, directly dialyzing glutathione S-transferase fusion CG11963 protein into CHO cells also shifts the dKCNQ G-V curve rightward. The effect of CG11963 persists in the presence of 1 mM adenosine triphosphate (ATP), a substrate of SCS. Taken together, our data define CG11963 as a new dKCNQ-binding protein capable of modulating the properties of the channel. Our evidence suggests that this modulation is mediated by direct interaction of CG11963 with the channel and is not dependent on ATP.